Mutations of the mitochondrial DNA (mtDNA) have been identified in several neuro-ophthalmological, neurological and, in neuromuscular diseases. However, the detailed molecular events associated with the pathology of these untreatable disorders are still poorly understood. These mutations occur in protein coding, tRNA or rRNA genes and have the common feature of impairing energy production in affected tissues. In the last three years we produced and characterized a large number of established cell lines containing different pathogenic mtDNA mutation, most of which affect tRNA genes. We propose to continue to characterize disease-related mtDNA alterations and use this collection of cell lines as a paradigm to study genotype-phenotype correlations. We will compare the activity of individual respiratory complexes, potential oxidative damage, and RNA and DNA metabolism in fourteen cell lines containing either wild-type mtDNA or one of nine different mtDNA alterations. We also propose to develop new approaches to ameliorate the phenotypic consequences of mtDNA mutations. This will be attempted by expressing modulatory genes into the nucleus of cells harboring pathogenic mtDNA mutations. These genes include: aminoacyl tRNA synthetase, re-coded mitochondrial genes and putative second-site functional suppressors currently under investigation.